PyMT-Maclow: A novel, inducible, murine model for determining the role of CD68 positive cells in breast tumor development

Abstract

CD68+ tumor-associated macrophages (TAMs) are pro-tumorigenic, pro-angiogenic and are associated with decreased survival rates in patients with cancer, including breast cancer. Non-specific models of macrophage ablation reduce the number of TAMs and limit the development of mammary tumors. However, the lack of specificity and side effects associated with these models compromise their reliability. We hypothesized that specific and controlled macrophage depletion would provide precise data on the effects of reducing TAM numbers on tumor development. In this study, the MacLow mouse model of doxycycline-inducible and selective CD68+ macrophage depletion was crossed with the murine mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) mouse model of spontaneous ductal breast adenocarcinoma to generate the PyMT-MacLow line. In doxycycline-treated PyMT-MacLow mice, macrophage numbers were decreased in areas surrounding tumors by 43%. Reducing the number of macrophages by this level delayed tumor progression, generated less proliferative tumors, decreased the vascularization of carcinomas and down-regulated the expression of many pro-angiogenic genes. These results demonstrate that depleting CD68+ macrophages in an inducible and selective manner delays the development of mammary tumors and that the PyMT-MacLow model is a useful and unique tool for studying the role of TAMs in breast cancer.

Fig 1. CD68+ and F4/80+ macrophages are analogous populations in PyMT tumors.

Tumors from PyMT mice were enzymatically digested and made into a single cell suspension. Cells were stained with antibodies against the surface markers CD45, CD11b and F4/80. Cells were then fixed, permeabilized and stained with anti-CD68. Fluorescence was measured by flow cytometry and the data was analyzed using Flowjo software. Dot plots shown were generated from CD45⁺ cells. CD68 expression was determined after gating on tumor-associated macrophages (CD45⁺CD11b⁺F4/80⁺), CD11b⁺F4/80^—cells and CD11b^—F4/80^—cells. Representative dot plots are shown from one of four mice analysed.

Fig 2. Doxycycline reduces the number of macrophages surrounding mammary tumors in PyMT-MacLow mice.

(A) Sections of mammary tissue were labelled for F4/80 (DAB brown cells) and counterstained with haematoxylin; a representative image at the early carcinoma stage of tumor development is shown for each genotype and treatment. (B) Images captured from slides scanned on an Aperio slide scanner were used to quantify the number of macrophages within (intratumoral) and on the perimeter of mammary tumors (peritumoral). The data was then grouped according to genotype and treatment group and the mean value +/- SD shown. Each individual data point represent the mean values for an individual tumor. Tumor samples were obtained from the following numbers of animals per treatment group the number of tumors analysed is indicated in brackets: PyMT UT = 5(27), PyMT Doxy = 5(33), PyMT-MacLow UT = 7(31), PyMT-MacLow Doxy = 7(39). Significance is from the SPSS nested analysis comparing data from doxycycline treated versus control animals for each tumor and genotype. NS = not significant, **P<0.01. Scalebar = 100 μm. (C) The percentage of animals that had any tumors of hyperplasia and/or Adenoma/mammary intraepithelial neoplasia stages (Adenoma/MIN) was calculated for each genotype and treatment group. A Chi-square test was used to calculate statistical significance.

Fig 3. The proliferative capacity of Adenoma/MIN tumors is reduced in macrophage deficient mice.

Mammary sections were labelled with an anti-Ki67 antibody as a marker of proliferation and counterstained with haematoxylin. (A) Images were captured from slides scanned on an Aperio slide scanner and split according to tumor grade, a representative image of an Adeno/MIN tumor (A/M) is shown for each genotype and treatment group. (B) The amount of Ki67 labelling in individual tumor areas (cells stained dark brown with DAB) was quantified and expressed as positivity using the Aperio positive pixel algorithm. Each data point on the graph represents the mean positivity for an individual tumor and the number of animals these tumors were taken from is shown below the x axis on each graph. The mean value ± SD was plotted and a nested analysis carried out in SPSS to compare data from doxycycline treated versus control animals from each tumor grade and genotype. ***P<0.001. Scalebar = 100 μm.

Fig 4. Loss of CD68⁺ macrophages negatively affects angiogenesis in PyMT mice.

Mammary sections were labelled with rabbit anti-mouse antibodies against CD31 (DAB brown) a marker of angiogenesis and counterstained with haematoxylin. (A) Images were captured from slides scanned on an Aperio slide scanner and a representative image of an early carcinoma is shown for both treatment groups. (B) Microvessel density (MVD) was expressed as cumulative chalkley score (CCS). Each point on the graph represents the CCS for individual early and late carcinomas obtained from the following numbers of animals: PyMT UT = 2(6), PyMT Doxy = 4(12), PyMT-MacLow UT = 3(11), PyMT-MacLow Doxy = 3(9), numbers in brackets correspond to the total number of carcinomas analysed for each group, the number of animals is also displayed on the graph below the axis. (C) Three samples of cDNA from mammary fat pads containing tumors were pooled for control and doxycycline treated PyMT-MacLow animals. Gene expression levels in the pooled cDNA samples were determined by hybridisation to two Mouse Angiogenesis RT² (Qiagen) plates (control and treated). Data was generated and interpreted in SDS 2.3 and any genes exhibiting a three-fold or greater change in the treated versus control group were considered biologically relevant and are shown on the graph. Scalebar = 100 μm. For part B nested analysis of the data was carried out in SPSS, **P<0.01.